Exogenous nitric oxide inhibits growth factor mediated angiogenesis by retinal vascular endothelial cells through a mechanism involving endogenous generation of superoxide

Abstract

Abstract: : Purpose: We have previously shown that EGF or FGF–mediated angiogenesis by retinal vascular endothelial cells (RVEC) is dependent upon the NO/cGMP pathway and that NO from exogenous sources, while pro–angiogenic alone, has a net anti–angiogenic outcome when applied in the presence of EGF or FGF. It was hypothesised that this paradox may be due to superoxide production by uncoupled endothelial nitric oxide synthase (NOS–3). Accordingly, antioxidants were tested for their ability to overcome the inhibitory effect of the NO donor SNAP on EGF–mediated angiogenesis in vitro. Direct inhibition of NOS–3 was also examined to ascertain whether such an oxidant–related anti–angiogenic effect was due to NOS–3 uncoupling. Methods: The antioxidants n–acetyl–cysteine (NAC), butylated hydroxytoluene (BHT) and peg–superoxidase dismutase (peg–SOD), and the NOS inhibitor L–NAME were tested individually in duplex 3–D Matrigel cultures of bovine RVEC in which a secondary gel layer containing the test substances (EGF+SNAP+antioxidant or L–NAME) was applied to a pre–formed endothelial tubular network within a primary Matrigel culture. The number of vascular sprouts invading the secondary layer was counted in 10 cultures per treatment and the results analysed using a one way ANOVA with a Tukey–Kramer post test for multiple comparisons. Results: As previously observed, exogenous NO inhibited EGF stimulated angiogenesis: Angiogenesis produced by EGF+SNAP was significantly less than EGF alone (p<0.001) or SNAP alone (p<0.001). Each of the antioxidants tested restored the angiogenic response to levels approaching that produced by EGF alone, with peg–SOD having the greatest effect (not significantly different to EGF alone: p>0.1). Interestingly, L–NAME had no effect on the NO–related inhibition of EGF–mediated angiogenesis (p>0.1), suggesting that NOS–3 was not the source of superoxide in this situation. Conclusions: Exogenous NO has an inhibitory effect on growth factor mediated angiogenesis by retinal vascular endothelial cells, an effect mediated by superoxide derived from an endothelial source other than NOS–3.

Keywords: vascular cells • nitric oxide • antioxidants